Center for Predictive Medicine for Biodefense and Emerging Infectious Diseases, University of Louisville, Louisville, KY, USA.
Virol J. 2013 Jan 10;10:19. doi: 10.1186/1743-422X-10-19.
Human respiratory syncytial virus (hRSV) is a highly contagious pathogen and is the most common cause of bronchiolitis and pneumonia for infants and children under one year of age. Worldwide, greater than 33 million children under five years of age are affected by hRSV resulting in three million hospitalizations and 200,000 deaths. However, severe lower respiratory tract disease may occur at any age, especially among the elderly or those with compromised cardiac, pulmonary, or immune systems. There is no vaccine commercially available. Existing therapies for the acute infection are ribavirin and the prophylactic humanized monoclonal antibody (Synagis® from MedImmune) that is limited to use in high risk pediatric patients. Thus, the discovery of new inhibitors for hRSV would be clinically beneficial.
We have developed and validated a 384-well cell-based, high-throughput assay that measures the cytopathic effect of hRSV (strain Long) in HEp-2 cells using a luminescent-based detection system for signal endpoint (Cell Titer Glo®). The assay is sensitive and robust, with Z factors greater than 0.8, signal to background greater than 35, and signal to noise greater than 24. Utilizing this assay, 313,816 compounds from the Molecular Libraries Small Molecule Repository were screened at 10 μM. We identified 7,583 compounds that showed greater than 22% CPE inhibition in the primary screen. The top 2,500 compounds were selected for confirmation screening and 409 compounds showed at least 50% inhibition of CPE and were considered active. We selected fifty-one compounds, based on potency, selectivity and chemical tractability, for further evaluation in dose response and secondary assays Several compounds had SI50 values greater than 3, while the most active compound displayed an SI50 value of 58.9.
A robust automated luminescent-based high throughput screen that measures the inhibition of hRSV-induced cytopathic effect in HEp-2 cells for the rapid identification of potential inhibitors from large compound libraries has been developed, optimized and validated. The active compounds identified in the screen represent different classes of molecules, including aryl sulfonylpyrrolidines which have not been previously identified as having anti-hRSV activity.
人类呼吸道合胞病毒（hRSV）是一种高度传染性病原体，是一岁以下婴儿和儿童毛细支气管炎和肺炎的最常见病因。在全球范围内，有超过 3300 万五岁以下儿童受到 hRSV 的影响，导致 300 万例住院和 20 万例死亡。然而，严重的下呼吸道疾病可能发生在任何年龄，尤其是老年人或心脏、肺部或免疫系统受损的人。目前尚无市售疫苗。现有的急性感染疗法是利巴韦林和预防性人源化单克隆抗体（来自 MedImmune 的 Synagis®），但仅限于高危儿科患者使用。因此，发现新的 hRSV 抑制剂将具有临床益处。
我们开发并验证了一种基于细胞的 384 孔高通量测定法，该测定法使用基于发光的终点检测系统（Cell Titer Glo®）测量 hRSV（Long 株）对 HEp-2 细胞的细胞病变效应。该测定法灵敏且稳健，Z 因子大于 0.8，信号与背景比大于 35，信号与噪声比大于 24。利用该测定法，从分子文库小分子库中筛选了 313816 种化合物，浓度为 10μM。我们在初筛中发现了 7583 种化合物，其对细胞病变效应的抑制率大于 22%。前 2500 种化合物被选为确证筛选，409 种化合物对细胞病变效应的抑制率大于 50%，被认为是活性化合物。我们根据效力、选择性和化学可处理性选择了 51 种化合物进行进一步的剂量反应和二次测定评估。一些化合物的 SI50 值大于 3，而最有效的化合物的 SI50 值为 58.9。
已开发、优化和验证了一种基于发光的自动化高通量筛选方法，该方法可测量 HEp-2 细胞中 hRSV 诱导的细胞病变效应的抑制，用于快速鉴定大型化合物库中的潜在抑制剂。筛选中发现的活性化合物代表不同类别的分子，包括芳基磺酰基吡咯烷，以前未被鉴定具有抗 hRSV 活性。
